The present invention relates generally to binding assays providing for determination of analytes in samples.
Assays directed to detection and quantification of physiologically significant materials in biological fluid and tissue samples are important tools in scientific research and in the health care field. Several different types of assay have been developed which are capable of detecting relatively highly concentrated components of common biological samples such as human serum. Such assays include high-resolution agarose gel electrophoresis and test procedures based on the catalytic activity of endogeneous enzymes. These methods generally do not have the sensitivity required to detect and quantify the numerous other physiologically important sample constituents which may be present at very low concentrations [e.g., endogeneous molecules intimately involved in cellular regulation (hormones, steroids, biochemical messengers); basic structural components of the organism (amino acids, proteins, polysaccharides); genetic material (DNA, RNA); vitamins, drugs and drug metabolites; toxins, pathogens and substances generated by the immune system].
The early biological assay techniques for the clinically important serum constituents such as the immuno-precipitation and immunodiffusion techniques developed in the 1940's also lacked the sensitivity necessary to detect and quantify most serum constituents of medical interest. In 1956 Berson and Yalow reported detection of soluble insulin-antibody complexes in the serum of insulin-treated diabetics injected with radiolabelled preparations of the serum hormone. See, Berson et al., J. CLIN. INVEST. 35: 170 (1956). The principles of this assay, commonly referred to as radioimmunoassay (RIA), were subsequently established and by the late 1960's the RIA was a major tool in endocrine laboratories. For example, virtually all the information now known about peptide hormonal physiology has resulted from the introduction of RIA and its ability to detect 10.sup.-10 to 10.sup.-12 molar concentrations of hormones.
The RIA assay technique was subsequently shown to be applicable to quantitative detection of any substance for which a specific antibody can be prepared, permitting development of a host of RIAs for chemical compounds such as drugs. In a broader sense the RIA principle has also been extended to systems in which other binding substances replace antibody, for example, in receptor assays. In 1980, the sales of immunodiagnostic reagents alone was estimated to be $229 million.
Although exhibiting the desired sensitivity, RIAs present several disadvantages inherent in the required reagents. The use of radioactive isotopes requires a special permit and a special laboratory. For this reason RIAs are performed by personnel separate from those in the routine clinical chemistry laboratory. Radiation can cause health hazards particularly for those working with the commonly used isotopes of iodine. In addition, the useful lifetime of the radiolabelled reagents employed is limited by half-life of the isotopes and the destructive processes that occur during isotopic decay. The equipment used to determine the amount of radioactivity in the samples is expensive and the counting of a series of samples is relatively time-consuming. (See, Smith et al., AMER. CLIN. BIOCHEM. 18: 253-74 (1981).) Overall, the amount of automation in the immunodiagnostic area is much less than that found in the routine clinical laboratory. Using an eight hour polyethylene glycol accelerated second antibody RIA for separation of free from bound antigen, only about 75-90 assays can be performed daily by a single technician employing manual pipettes and a single channel gamma counter.
To overcome the problems associated with RIA, immunoassay techniques employing nonisotopic labels have been developed. These nonisotopic assays, referred to as enzyme-linked immunoabsorbent assays (ELISA), fluoroimmunoassays (FIA), and luminescent immunoassay (LIA), according to the label employed, avoid many of the problems associated with RIAs and possess sensitivities near to that of the RIA. More recently, enzyme-linked assays have become increasingly popular and are replacing RIAs in many cases due to their more simple protocol relative to RIAs. As many as 2,000 assays per day can be run by a technician employing a solid-phase ELISA in microtiter plates with manual pipettes. These types of assays also have permitted the development of "homogeneous" immunoassays in which the bound and free labelled material need not be separated prior to the detection and measurement step. The RIA procedure requires the separation of free from bound labelled material for estimation of analyte concentration, a "heterogeneous" system. Sensitive assays in which antigen-antibody reactions could be detected without separation of free from complexed antigen are also more simple to automate.
Although superior to RIAs in several respects, the nonisotopic assays described above also exhibit problems caused by endogeneous interfering factors present in the reaction mixture. Proteins and other components commonly found in serum samples may exhibit fluorescent, chemiluminescent and enzymatic activity similar to that of the employed label. In addition, the activity of these labels may be inhibited by the presence of endogeneous compounds which absorb or scatter the emitted light of photophore-labels, similarly color compounds relative to chromophore labels, and catalytic enzymes which degrade enzyme labels. Determination of the activity of the employed label may also be impaired by the turbidity of the sample as in the case of whole blood samples. To a certain extent, these problems may be minimized by assay techniques employing a separation step wherein bound labelled material is separated from the sample, washed with buffer and the label activity is thereafter determined or wherein the separation of bound and unbound labelled material is achieved by partitioning them between immiscible aqueous phases. See, Mattiasson et al., ADVANCES IN APPLIED MICROBIOLOGY 28: 117-47 (1981); U.S. Pat. No. 4,312,944. However, the potential still exists that serum interfering components may be present. The determination step often requires use of photodetectors and other complex instrumentation generally available only in clinical laboratories.
There continues to exist in the art, therefore, a need for nonisotopic binding assays for determination of the presence of analytes which more rigorously avoid problems of interference inherent in detection and measurement steps of the assay and which avoid the need for sophisticated instrumentation while providing high sensitivity detection by methods which make them available for use in physicians' offices or elsewhere outside the clinical laboratory.